The present invention relates to pseudomycin natural products including pseudomycins Axe2x80x2 and Bxe2x80x2, methods for making such pseudomycins, and methods employing antifungal activity of these pseudomycins.
Fungal infections are a significant cause of disease, degradation of quality of life, and mortality among humans, particularly for immune compromised patients. The incidence in fungal infections in humans has increased greatly in the past 20 years. This is in part due to increased numbers of people with immune systems weakened or devastated by organ transplants, cancer chemotherapy, AIDS, age, and other similar disorders or conditions. Such patients are prone to attack by fungal pathogens that are prevalent throughout the population but are kept in check by a functioning immune system. These pathogens are difficult to control because some existing antifungal agents are either highly toxic or only inhibit fungal activity. For example, the polyenes are fungicidal but toxic; whereas, the azoles are much less toxic but only fungistatic. More importantly, there have been recent reports of azole and polyene resistant strains of Candida which severely limits therapy options against such strains.
Pseudomonas syringae produce several classes of antifungal or antibiotic agents, such as the pseudomycins, syringomycins, syringotoxins, and syringostatins, which are lipodepsinonapeptides. Natural strains and transposon generated mutants of P. syringae produce these lipodepsinonapeptides. Several of the pseudomycins, syringomycins and other lipodepsipeptide antifungal agents have been isolated, chemically characterized, and shown to possess wide spectrum antifungal activity, including activity against important fungal pathogens in both humans and plants. For example, pseudomycins A, B, C and Cxe2x80x2 have each been isolated and purified and their structures have been characterized by methods including amino acid sequencing, NMR, and mass spectrometry. See. e.g. Ballio et al., xe2x80x9cNovel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins,xe2x80x9d FEBS Lett. 355. 96-100 (1994) and U.S. Pat. No. 5,576,298. The pseudomycins, the syringomycins, the syringotoxins, and the syringostatins represent structurally distinct families of antifungal compounds.
None of the pseudomycins, syringomycins, syringotoxins, or syringostatins has been brought to market for antifungal therapy. Discovery of undesirable side effects, making formulations, scaling up production, and other development problems have thus far prevented exploitation of the pseudomycins, syringomycins, syringotoxins, or syringostatins against the full range of fungal infections that affect animals, humans and plants. There remains a need for an antifungal agent that can be used against infections not treated by existing antifungal agents and for application against infections in animals, humans, or plants.
The present invention provides a pseudomycin natural product produced by P. syringae. The pseudomycin natural product includes a depsinonapeptide ring with the sequence Ser-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr, more specifically, L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl), with the carboxyl group of the ClThr and the hydroxyl group of the serine closing the ring with a lactone bond. Pseudomycin Axe2x80x2 (IA) includes a 3,4-dihydroxypentadecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine. 
Pseudomycin Bxe2x80x2 (IB) includes a 3-hydroxydodecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine. 
The invention also relates to methods employing a pseudomycin natural product, such as pseudomycin Axe2x80x2, pseudomycin Bxe2x80x2 or a mixture thereof, for inhibiting fungal activity or for reducing the symptoms of a fungal infection in a patient in need thereof. Such methods can kill the fungus, decrease the burden of a fungal infection, reduce fever and/or increase the general well being of a patient. The methods of the invention are effective against fungi such as Candida parapsilosis, Candida albicans, Cryptococcus neoformans, and/or Histoplasma capsulatum. 
Pseudomycins
As used herein, pseudomycin or pseudomycin natural product refers to one or more members of a family of antifungal agents that has been isolated from the bacterium Pseudomonas syringae. A pseudomycin is a lipodepsipeptide, a cyclic peptide including one or more unusual amino acids and having one or more appended hydrophobic or fatty acid side chains. Specifically, the pseudomycins are lipodepsinonapeptides, with a cyclic peptide portion closed by a lactone bond and including the unusual amino acids 4-chlorothreonine, 3-hydroxyaspartic acid, dehydro-2-aminobutyric acid, and 2,4-diaminobutyric acid. It is believed that these unusual amino acids are involved in biological characteristics of the pseudomycins, such as stability in serum and their killing action.
Each pseudomycin has the same cyclic peptide nucleus, but they differ in the hydrophobic side chain attached to this nucleus. Each pseudomycin has a cyclic nonapeptide ring having the sequence Ser-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr (i.e., Serine; 2,4-Diaminobutyric acid; Aspartic acid; Lysine; 2,4-Diaminobutyric acid; alloThreonine; Dehydro-2-aminobutyric acid; 3-hydroxyAspartic acid; 4-chloroThreonine), with the carboxyl group of the ClThr and the hydroxyl group of the serine closing the ring with a lactone bond. The lipophilic moiety is attached to the amine group of the N-terminal serine. The amine group of the serine forms an amide bond with the carboxyl of a 3,4-dihydroxytetradecanoyl moiety in pseudomycin A, a 3-monohydroxytetradecanoyl moiety in pseudomycin B, a 3,4-dihydroxyhexadecanoyl moiety in pseudomycin C and a 3-monohydroxyhexadecanoyl moiety in pseudomycin Cxe2x80x2. The carboxyl group of the serine forms an amide bond with the Dab of the ring.
Pseudomycins Axe2x80x2 and Bxe2x80x2
As used herein the terms pseudomycin Axe2x80x2 and pseudomycin Bxe2x80x2 refer to antifungal agents that have been isolated from the bacterium Pseudomonas syringae. Pseudomycins Axe2x80x2 and Bxe2x80x2 are pseudomycins having the characteristic depsinonapeptide ring with the sequence Ser-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr, with the carboxyl group of the ClThr and the hydroxyl group of the serine closing the ring with a lactone bond. Pseudomycin Axe2x80x2 includes a 3,4-dihydroxypentadecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine. Pseudomycin Bxe2x80x2 includes a 3-hydroxydodecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine.
Biological Activities of Pseudomycins
A pseudomycin has several biological activities including killing various fungi, such as fungal pathogens of plants and animals. In particular, a pseudomycin is an active antimycotic agent against fungi that cause opportunistic infections in immune compromised individuals. These fungi include various species of Candida including C. parapsilosis, C. albicans, C. glabrata, C. tropicalis, and C. krusei. They also incldue other genera such as Cryptococcus neoformans, Aspergillus fumigatus, and Histoplasma capsulatum. Killing, rather than inhibiting the growth of fungi, particularly of fungal pathogens, is a desirable and preferred biological activity of an antifungal, such as pseudomycin Axe2x80x2 and/or Bxe2x80x2.
The pseudomycins have been shown to be toxic to a broad range of plant-pathogenic fungi including Rynchosporium secalis, Ceratocystis ulmi, Rizoctonic solani, Sclerotinia sclerotiorum, Verticillium albo-atrum, Verticillium dahliae, Thielaviopis basicola, Fusarium oxysporum and Fusarium culmorum. (see Harrison, L., et al., xe2x80x9cPseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity,xe2x80x9d J. of General Microbiology, 7, 2857-2865 (1991).) In addition, P. syringae MSU 16H has been shown to confer a greater protection than the wild-type strain in elms infected with Ceratocystic ulmi, the causal agent of Dutch elm disease. (see e.g., Lam et al. Proc. Natl. Sci. USA, 84, 6447-6451 (1987)).
Pseudomonas syringae 
Pseudomonas syringae include a wide range of bacteria that are generally associated with plants. Some of the P. syringae are plant pathogens, while others are only weakly pathogenic or are saprophytes. Many different isolates of P. syringae produce one or more cytotoxic agents that can help this bacterium survive in the wild where it must compete with fungi and other bacteria. The cytotoxic agents produced by P. syringae include anti-fungal agents such as the pseudomycins, the syringomycins, the syringotoxins, and the syringostatins.
Strains of P. syringae that produce one or more pseudomycins have been described in the art. For example, wild type strain MSU 174 (isolated from a Montana barley field) and a mutant of this strain generated by transposon mutagenesis using TN905 (MSU 16H) are described in U.S. Pat. No. 5,576,298, issued Nov. 19, 1996 to G. Strobel et al.; Harrison et al., J. xe2x80x9cPseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity,xe2x80x9d Gen. Microbiology 137, 2857-2865 (1991); and Lamb et al., xe2x80x9cTransposon mutagenesis and tagging of fluorescent pseudomonas: Antimycotic production is necessary for control of Dutch elm disease,xe2x80x9d Proc. Natl. Acad. Sci. USA 84, 6447-6451 (1987). Methods for growth of various strains of P. syringae and their use in production of antifungal agents such as pseudomycins are also disclosed in U.S. patent application Ser. No. 09/958,996 by Matthew D. Hilton et al. entitled xe2x80x9cPseudomycin Production By Pseudomonas Syringaexe2x80x9d submitted evendate herewith and described below. Cultures of MSU 174 and MSU 16H are on deposit at Montana State University (Bozeman, Mont., USA) and available from the American Type Culture Collection (Parklawn Drive, Rockville, Md., USA). The disclosures of the references cited in this paragraph are incorporated herein by reference.
The present invention includes a strain, an isolate, and a biologically-purified culture of P. syringae that produces pseudomycin Axe2x80x2 and/or Bxe2x80x2, in amounts at least about 10 xcexcg/mL. Preferably, the biologically-purified culture of a microorganism is of Pseudomonas syringae strain MSU 16H, 25-B1, 67H1, or 7H9-1, or a mutant, variant, isolate, or recombinant of these strains that produces pseudomycin Axe2x80x2 and/or Bxe2x80x2. Cultures MSU 174 and MSU 16H were obtained as described in the references cited herein above.
A strain of P. syringae that is suitable for production of pseudomycin Axe2x80x2 and/or Bxe2x80x2 can be isolated from environmental sources including plants, such as barley plants, citrus plants, and lilac plants, and also from sources such as soil, water, air, and dust. A preferred strain is isolated from plants. Strains of P. syringae that are isolated from environmental sources can be referred to as wild type. As used herein, xe2x80x9cwild typexe2x80x9d refers to a dominant genotype which naturally occurs in the normal population of P. syringae (i.e., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation). As is the case with other organisms, the characteristics of the pseudomycin Axe2x80x2 and/or Bxe2x80x2 producing cultures employed in this invention, P. syringae strains such as MSU 174, MSU 16H, MSU 206, 25-B1, 7H9-1, and 67 H1 are subject to variation. Thus, progeny of these strains, e.g., recombinants, mutants and variants, may be obtained by methods well-known to those skilled in the art.
Mutant strains of P. syringae are also suitable for production of pseudomycin Axe2x80x2 and/or Bxe2x80x2. As used herein, mutant refers to a sudden heritable change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, including radiation and various chemicals. Mutant P. syringae of the present invention can be produced using a variety of mutagenic agents including radiation such as ultraviolet light, x-rays; chemical mutagens, site-specific mutagenesis, and transposon mediated mutagenesis. Examples of chemical mutagens are ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-Nxe2x80x2-nitrosoguanine (NTG), and nitrous acid.
Pseudomycin Axe2x80x2 and/or Bxe2x80x2 producing mutants of P. syringae of the present invention can be produced by treating the bacteria with an amount of a mutagenic agent effective to produce mutants that overproduce pseudomycin Axe2x80x2 and/or Bxe2x80x2, that produce pseudomycin Axe2x80x2 and/or Bxe2x80x2 in excess over other pseudomycins, or that produce pseudomycin Axe2x80x2 and/or Bxe2x80x2 under advantageous growth conditions. While the type and amount of mutagenic agent to be used can vary, a preferred method is to serially dilute NTG to levels ranging from 1 to 100 xcexcg/ml. Preferred mutants of the invention are those that overproduce pseudomycin Axe2x80x2 and/or Bxe2x80x2 grow in minimal defined media. The mutants overproduce pseudomycin Axe2x80x2 and/or Bxe2x80x2 preferably to at least about 10 xcexcg/mL.
Environmental isolates, mutant strains, and other desirable strains of P. syringae can be subjected to selection for desirable traits of growth habit, growth medium, nutrient source, carbon source, growth conditions, and amino acid requirements. Preferably, a pseudomycin Axe2x80x2 and/or Bxe2x80x2 producing strain of P. syringae is selected for growth on minimal defined medium, such as N21 medium, and/or for production pseudomycin Axe2x80x2 and/or Bxe2x80x2 at levels greater than about 10 xcexcg/mL. Preferred strains exhibit the characteristic of producing pseudomycin Axe2x80x2 and/or Bxe2x80x2 when grown on a medium including glycine and, optionally, either a lipid, a potato product, or a combination thereof.
Recombinant strains can be developed by transforming the P. syringae strains, using established laboratory procedures well-known to those skilled in the art. Through the use of recombinant technology, the P. syringae strains can be transformed to express a variety of gene products in addition to the antibiotics these strains produce. For instance, one can transform the strains with a recombinant vector that confers resistance to an antibiotic to which the strains are normally sensitive. Transformants thus obtained will produce not only pseudomycins, such as pseudomycins Axe2x80x2 and/or Bxe2x80x2, but also the resistance-conferring enzyme that allows selection of the transformed from wild-type cells. Furthermore, using similar techniques, one can modify the present strains to introduce multiple copies of the endogenous pseudomycin-biosynthesis genes to achieve greater pseudomycin, such as pseudomycin Axe2x80x2 and/or Bxe2x80x2 yield. Progeny, i.e. natural and induced variants, mutants and recombinants, of the P. syringae strains 25-B1, 67H1, and 7H9-1 which retain the characteristic of pseudomycin, such as pseudomycin Axe2x80x2 and/or Bxe2x80x2 overproduction are part of this invention.
Growth of Pseudomonas syringae 
As described herein, xe2x80x9caqueous nutrient mediaxe2x80x9d refers to a water-base composition including minerals and organic compounds and their salts necessary for growth of the bacterium used in the present invention. Preferred nutrient media contain an effective amount of three or fewer amino acids, preferably, glutamic acid, glycine, histidine or a combination thereof. In one embodiment, the medium contains an effective amount of glycine and, optionally, one or more of a potato product and a lipid. Glycine can be provided as a single amino acid or as part of a mixture of amino acids, such as hydrolyzed protein. Suitable lipids include soybean oil, or a fatty acid. Suitable potato products include potato dextrose broth, potato dextrin, potato protein, and commercial mashed potato mix food product. Preferred minerals in the nutrient medium include salt mixtures typically used in cell culture and fermentation, such as Czapek mineral salts solution (e.g., KCl, MgSO4, and FeSO4). The organic compound in the nutrient media preferably includes glucose and can optionally include soluble starch; other like organic compounds can also be included. The pH of the medium is preferably between about 4 and 6.5, more preferably about 4.5 to about 5.7, most preferably about 5.2.
Although the amount of each ingredient in the nutrient broth is not typically critical to growth of the bacteria or to production of pseudomycin Axe2x80x2 and/or Bxe2x80x2 certain levels of nutrients are advantageous. A preferred amount of glycine is about 0.1 g/L to about 10 g/L, more preferably about 0.3 g/L to about 3 g/L, most preferably about 1 g/L. A preferred amount of lipid is about 1 g/L to about 10 g/L of an oil product such as soybean oil, more preferably about 0.5 g/L to about 2 g/L of soybean oil. A preferred amount of a fatty acid or fatty acid ester is about 0.5 g/L to about 5 g/L. Preferred amounts of potato products include about 12 g/L to about 36 g/L, preferably about 24 g/L of potato dextrose broth; about 5 g/L to about 50 g/L, preferably about 30 g/L of commercial mashed potato mix; about 1 g/L to about 30 g/L, preferably about 20 g/L of potato dextrin; or about 1 g/L to about 10 g/L, preferably about 4 g/L of potato protein. A preferred nutrient medium includes minerals, preferably, KCl at about 0.02 to about 2 g/L, more preferably about 0.2 g/L; MgSO4, preferably MgSO4.7H2O, at about 0.02 to about 2 g/L, more preferably about 0.2 g/L; and FeSO4, preferably FeSO4.7H2O, at about 0.4 to about 40 mg/L, more preferably about 4 mg/L. When present, soluble starch is preferably at about 0.5 to about 50 g/L, more preferably about 5 g/L. Glucose is preferably present at about 2 to about 80 g/L, more preferably about 20 g/L.
P. syringae are typically grown in the media described under conditions of controlled or regulated pH, and temperature. P. syringae grow and pseudomycin Axe2x80x2 and/or Bxe2x80x2 at temperatures between about 15xc2x0 C. and about 35xc2x0 C., preferably about 20xc2x0 C. to about 30xc2x0 C., more preferably about 22xc2x0 C. to about 27xc2x0 C., most preferably about 25xc2x0 C. P. syringae grow and produce pseudomycin Axe2x80x2 and/or Bxe2x80x2 at pH between about 4 and about 9, preferably about 4 and about 6, more preferably about 4.5 to about 5.5. Typically growth of P. syringae does not occur when the temperature is above 37xc2x0 C. or below 10xc2x0 C. or when the pH is above 9 or below 4.
Method for Production of Pseudomycins Axe2x80x2 and Bxe2x80x2
To produce pseudomycin Axe2x80x2 and/or Bxe2x80x2 from a wild type or mutant strain of P. syringae, the organism is cultured with agitation in an aqueous nutrient medium including an effective amount of three or fewer amino acids. The three or fewer amino acids are preferably glutamnic acid, glycine, histidine, or a combination thereof. In one preferred embodiment, the amino acids include glycine and, optionally, one or more of a potato product and a lipid. Culturing is conducted under conditions effective for growth of P. syringae and production of pseudomycin Axe2x80x2 and/or Bxe2x80x2. Effective conditions include temperature of about 22xc2x0 C. to about 27xc2x0 C., and a duration of about 36 hours to about 96 hours. When cultivated on the media such as those described herein, P. syringae can grow in cell densities up to about 10-15 g/L dry weight and produce pseudomycins Axe2x80x2 and/or Bxe2x80x2 in total amounts at least about 10 xcexcg/mL.
Controlling the concentration of oxygen in the medium during culturing of P. syringae is advantageous for production of pseudomycin Axe2x80x2 and/or Bxe2x80x2. Preferably, oxygen levels are maintained at about 5% to about 50% saturation, preferably about 30% saturation. Sparging with air, with pure oxygen, or with gas mixtures including oxygen can regulate the concentration of oxygen in the medium. Further, adjustment of the agitation rate can be used to adjust the oxygen transfer rate.
Controlling the pH of the medium during culturing of P. syringae is advantageous for production of a pseudomycin Axe2x80x2 and/or Bxe2x80x2. Pseudomycins, such as pseudomycins Axe2x80x2 and/or Bxe2x80x2, are labile at basic pH, and significant degradation can occur if the pH of the culture medium is above about 6 for more than about 12 hours. Preferably, the pH of the culture medium is maintained at less than about 6, preferably less than about 5.5, and preferably above 4.0. The pH is preferably maintained at about 5 to about 5.4, more preferably about 5.0 to about 5.2. Although not limiting to the present invention, it is believed that pseudomycin degradation at basic pH is due to opening of the lactone ring and conversion of ClThr to Thr.
P. syringae can produce pseudomycins Axe2x80x2 and/or Bxe2x80x2 when grown in batch culture. However, fed-batch or semi-continuous feed of glucose and, optionally, an acid or base, such as ammonium hydroxide, to control pH, enhances pseudomycin production. Pseudomycin production by P. syringae can be further enhanced by using continuous culture methods in which glucose and, optionally, an acid or base, such as ammonium hydroxide, to control pH, are fed automatically.
Pseudomycins Axe2x80x2 and/or Bxe2x80x2 can be detected, determined, isolated, and/or purified by any of a variety of methods known to those of skill in the art. For example, the level of pseudomycin activity in a broth or in an isolated or purified composition can be determined by antifungal action against a fungus such as Candida. Numerous methods are known for the preparation and analysis of the pseudomycins. For example, one or more pseudomycins can be isolated and purified by chromatography, such as HPLC.
Pharmaceutical Uses
Formulations and Antifungal Action of Pseudomycin Axe2x80x2 or Bxe2x80x2
Each of pseudomycin Axe2x80x2 and Bxe2x80x2 show in vitro and in vivo activity and therefore may useful in combating either systemic fungal infections or fungal skin infections. Accordingly, the present invention provides a method of inhibiting fungal activity including contacting pseudomycin Axe2x80x2 and/or Bxe2x80x2 or a pharmaceutically acceptable salt thereof with a fungus. A preferred method includes inhibiting growth or activity of various fungi including C. parapsilosis, C. albicans, Cryptococcus neoformans, and Histoplasma capsulatum. As used herein contacting a compound of the invention with a parasite or fungus refers to a union or junction, or apparent touching or mutual tangency of a compound of the invention with a parasite or fungus. However, the term contacting does not imply any mechanism of inhibition.
The present invention further provides a method of treating a fungal infection which includes administering an effective amount of pseudomycin Axe2x80x2 and/or Bxe2x80x2, or a pharmaceutically acceptable salt, hydrate, or ester thereof, to a host in need of such treatment. A preferred method includes treating an infection by various fungi including C. parapsilosis, C. albicans, Cryptococcus neoformans, and Histoplasma capsulatum. When administered in an effective and appropirate amount, a formulation of pseudomycin Axe2x80x2 and/or Bxe2x80x2 reduces the burden of a fungal infection, reduces symptoms associated with the fungal infection, and can result in the elimination of the fungal infection.
Some patients in need of antifungal therapy have severe symptoms of infection, such as high fever, and are likely to be in intensive or critical care. Various fungi can cause such serious infections. Candida spp., for example, may cause mucosal and serious systemic infections. Azole and polyene resistant strains of Candida have been reported with increasing frequency. Aspergillus causes life-threatening systemic infections. Cryptococcus is responsible for meningitis. Such serious fungal infections may occur in immune compromised patients, such as those receiving organ or bone marrow transplants, undergoing chemotherapy for cancer, recovering from major surgery, or suffering from HIV infection. For such patients, antifungal therapy would typically include intravenous administration of a formulation containing pseudomycin Axe2x80x2 and/or Bxe2x80x2 over several days or more to halt the infection.
With respect to antifungal activity, the term xe2x80x9ceffective amount,xe2x80x9d means an amount of a compound of the present invention which is capable of inhibiting fungal growth or activity, or reducing symptoms of the fungal infection. For most fungal infections reduction of symptoms of the infection includes reduction of fever, return to consciousness, and increased well being of the patient. Preferably, symptoms are reduced by killing the fungus to eliminate the infection or to bring the infection to a level tolerated by the patient or controlled by the patient""s immune system. As used herein inhibiting refers to inhibiting fungal activity, including stopping, retarding or prophylactically hindering or preventing the growth or any attending characteristics and results from the existence of a fungus.
The dose administered will vary depending on such factors as the nature and severity of the infection, the age and general health of the host and the tolerance of the host to the antifungal agent. Typically, the compositions will be administered to a patient (human or other animal, including mammals such as, but not limited to, cats, horses and cattle and avian species) in need thereof, in an effective amount to inhibit the fungal infection. The particular dose regimen likewise may vary according to such factors and may be given in a single daily dose or in-multiple doses during the day. The regimen may last from about 2-3 days to about 2-3 weeks or longer. A typical daily dose (administered in single or divided doses) will contain a dosage level of from about 0.01 mg/kg to about 100 mg/kg of body weight of an active compound of this invention. Preferred daily doses generally will be from about 0.1 mg/kg to about 60 mg/kg and ideally from about 2.5 mg/kg to about 40 mg/kg. For serious infections, the compound can be administered by intravenous infusion using, for example, 0.01 to 10 mg/kg/hr of the active ingredient.
The present invention also provides pharmaceutical formulations useful for administering the antifungal compounds of the invention. Accordingly, the present invention also provides a pharmaceutical formulation including one or more pharmaceutically acceptable carriers, diluents, vehicles, excipients, or other additives and pseudomycin Axe2x80x2 and/or Bxe2x80x2. The active ingredient in such formulations includes from 0.1% to 99.9% by weight of the formulation, more generally from about 10% to about 30% by weight. By xe2x80x9cpharmaceutically acceptablexe2x80x9d it is meant that the carrier, diluent or excipient is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The formulation can include additives such as various oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, and sesame oil. Suitable pharmaceutical excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The compositions can be subjected to conventional pharmaceutical expedients, such as sterilization, and can contain conventional pharmaceutical additives, such as preservatives, stabilizing agents, wetting, or emulsifying agents, salts for adjusting osmotic pressure, and buffers. Suitable pharmaceutical carriers and their formulations are described in Martin, xe2x80x9cRemington""s Pharmaceutical Sciences,xe2x80x9d 15th Ed.; Mack Publishing Co., Easton (1975), see, e.g., pp. 1405-1412 and pp. 1461-1487.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d, as used herein, refers to salts of the compounds described above that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base. Such salts are known as acid addition and base addition salts.
Acids commonly employed to form acid addition salts are mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Examples of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, napththalene-2-sulfonate, and mandelate. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, and bicarbonates. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, and calcium carbonate. The potassium and sodium salt forms are particularly preferred.
It should be recognized that the particular counterion forming a part of any salt of this invention is not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
Pseudomycin Axe2x80x2 and/or Bxe2x80x2 may be administered parenterally, for example using intramuscular, subcutaneous, or intraperitoneal injection, nasal, or oral routes. In addition to these methods of administration, pseudomycin Axe2x80x2 and/or Bxe2x80x2 may be applied topically for superficial skin infections, or eradication or inhibition of fungi in the mucus.
For parenteral administration the formulation includes pseudomycin Axe2x80x2 and/or Bxe2x80x2 and a physiologically acceptable diluent such as deionized water, physiological saline, 5% dextrose and other commonly used diluents. The formulation may contain a cyclodextrin and/or a solubilizing agent such as a polyethylene glycol or polypropylene glycol or other known solubilizing agent. Such formulations may be made up in sterile vials containing the antifungal and excipient in a dry powder or lyophilized powder form. Prior to use, a physiologically acceptable diluent is added and the solution withdrawn via syringe for administration to the patient.
The present pharmaceutical formulations are prepared by known procedures using known and readily available ingredients. In making the compositions of the present invention, the active ingredient will generally be admixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
For oral administration, the antifungal compound is filled into gelatin capsules or formed into tablets. Such tablets may also contain a binding agent, a dispersant or other suitable excipients suitable for preparing a proper size tablet for the dosage pseudomycin Axe2x80x2 and/or Bxe2x80x2. For pediatric or geriatric use the antifungal compound may be formulated into a flavored liquid suspension, solution or emulsion. A preferred oral formulation is linoleic acid, cremophor RH-60 and water and preferably in the amount (by volume) of 8% linoleic acid, 5% cremophor RH-60, 87% sterile water and pseudomycin Axe2x80x2 and/or Bxe2x80x2 in an amount of from about 2.5 to about 40 mg/ml.
For topical use the antifungal compound may be formulated with a dry powder for application to the skin surface or it may be formulated in a liquid formulation including a solubilizing aqueous liquid or non-aqueous liquid, e.g., an alcohol or glycol.
Uses of Formulations of Pseudomycin Axe2x80x2 or Bxe2x80x2
The present invention also encompasses a kit including the present pharmaceutical compositions and to be used with the methods of the present invention. The kit can contain a vial which contains a formulation of the present invention and suitable carriers, either dried or in liquid form. The kit further includes instructions in the form of a label on the vial and/or in the form of an insert included in a box in which the vial is packaged, for the use and administration of the compounds. The instructions can also be printed on the box in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow a worker in the field to administer the drug. It is anticipated that a worker in the field encompasses any doctor, nurse, or technician who might administer the drug.
The present invention also relates to a pharmaceutical composition including a formulation of pseudomycin Axe2x80x2 and/or Bxe2x80x2 and that is suitable for administration by injection. According to the invention, a formulation of pseudomycin Axe2x80x2 and/or Bxe2x80x2, can be used for manufacturing a composition or medicament suitable for administration by injection. The invention also relates to methods for manufacturing compositions including a formulation of pseudomycin Axe2x80x2 and/or Bxe2x80x2 in a form that is suitable for administration by injection. For example, a liquid or solid formulation can be manufactured in several ways, using conventional techniques. A liquid formulation can be manufactured by dissolving pseudomycin Axe2x80x2 and/or Bxe2x80x2, in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients.
Agricultural Uses
Antibiotics produced from P. syringae NRRL B-12050 have been demonstrated to effectively treat Dutch elm disease, (see, e.g., U.S. Pat. Nos. 4,342,746 and 4,277,462). In particular, P. syringae MSU 16H has been shown to confer a greater protection than the wild-type strain in elms infected with Ceratocystis ulmi, the causal agent of Dutch elm disease, (see e.g., Lam et al. Proc. Natl. Sci. USA, 84, 6447-6451 (1987)). More extensive tests on field-grown elms confirmed the phenomenon of biocontrol at the prophylactic level. Hence, the pseudomycins of the present invention may be useful as a preventative treatment for Dutch Elm disease. The pseudomycins have been shown to be toxic to a broad range of plant-pathogenic fungi including Rynchosporium secalis, Ceratocystis ulmi, Rizoctonia solani, Sclerotinia sclerotiorum, Verticillium albo-atrum, Verticillium dahliae, Thielaviopis basicola, Fusarium oxysporum and Fusarium culmorum, (see Harrison, L., et al., xe2x80x9cPseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity,xe2x80x9d J. General Microbiology, 7, 2857-2865 (1991).) Consequently, the isolated pseudomycin Axe2x80x2 and/or Bxe2x80x2 (including hydrates, solvates, and esters thereof) may be useful in the treatment of fungi in plants (in particular, V. albo-atrum, Rhizoctonia solani and F. oxysporum) either as a direct treatment or preventative treatment. Generally, the infected plants are treated by injecting or spraying an aqueous suspension of the pseudomycin compounds into or onto the plant. Means of injection are well-known to those skilled in the art (e.g., gouge pistol). Any means of spraying the suspension may be used that distributes an effective amount of the active material onto the plant surface. The suspension may include other additives generally used by those skilled in the art, such as solubilizers, stabilizers, wetting agents, and combinations thereof.
Treatment of the plant may also be accomplished using a dry composition containing the isolated pseudomycin Axe2x80x2 and/or Bxe2x80x2 compounds. The dry formulation may be applied to the plant surface by any means well-known to those skilled in the art, such as spraying or shaking from a container.